Buprenorphine dimer and its use in treatment of gastrointestinal disorders

ABSTRACT

The present invention provides a buprenorphine dimer compound, wherein the two buprenorphine portions are linked via an ethylene spacer, wherein the spacer is bonded to the two opioid molecules via an ether bond. Pharmaceutical compositions comprising such a buprenorphine dimer drug are also disclosed, and the use of such compounds in the treatment of gastrointestinal hyperalgesia generally and in particular diarrhea-predominant irritable bowel syndrome.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Application Ser. No. 61/985,207, filed Apr. 28,2014; U.S. Provisional Application Ser. No. 62/101,768, filed Jan. 9,2015; and U.S. Provisional Application Ser. No. 62/176,883, filed Jan.9, 2015; the disclosures of each being incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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BACKGROUND OF THE INVENTION Buprenorphine

Buprenorphine is a semi-synthetic, mixed μ agonist-κ-antagonist opioidreceptor modulator that is used to treat opioid addiction in higherdosages, to control moderate acute pain in non-opioid-tolerantindividuals in lower dosages and to control moderate chronic pain ineven smaller doses. Its structure is:

Buprenorphine is currently indicated for the treatment of pain asintravenous, sublingual and transdermal dosage forms. Buprenorphine isalso indicated for the treatment of opiate addiction. Althoughbuprenorphine has a long half-life and can be administered once daily,the oral bioavailability of buprenorphine is very low due to extensivepresystemic extraction. Consequently sublingual administration ofbuprenorphine is required to achieve clinically effective systemicplasma concentrations. Even with sublingual administration,buprenorphine is only about 30% available to systemic circulation.

Diarrhea-Predominant Irritable Bowel Syndrome

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a highlyprevalent gastrointestinal disorder that is often accompanied, inaddition to diarrhea, by both visceral and somatic hyperalgesia(enhanced pain from colorectal and somatic stimuli), discomfort,bloating, and gas.

According to the International Foundation for FunctionalGastrointestinal Disorders, IBS-D is estimated to affect between 25-45million Americans. It is the most common diagnosis made bygastroenterologists, and is one of the disorders most frequently treatedby primary care physicians.

Irritable bowel syndrome has a very heavy impact on the quality of lifeand has high social costs. The disease has a fluctuating trend, buttends to be chronic or subchronic. Although there is no evidence thatthe presence of IBS involves deterioration in patient life expectancy,it significantly reduces health-related quality of life and workproductivity. In the more severe cases patients may experience severalepisodes of abdominal pain and diarrhea per day resulting in severeimpairment of relationships and in the workplace.

IBS-D Treatments

Bile acid binders, amitryptyline, probiotics, mast cell stabilizers and5-ASA have been used off-label in the treatment of IBS-D, albeit withoutcompelling evidence of chronic efficacy. The anti-diarrheal loperamide,a synthetic opioid, has been used similarly, but its uninhibited fullopioid agonist activity often results in severe constipation.

Among drugs in development for IBS-D is LX 1033, an inhibitor ofserotonin synthesis in the gastro-intestinal tract, currently beingdeveloped by Lexicon Pharmaceuticals. Its mechanism of action, however,does not support pain alleviation. DNK-333 (Novartis), a neurokininantagonist, was withdrawn from study for IBS-D following Phase IIstudies for want of efficacy. Ibodutant (Menarini), another neurokininantagonist in Phase II trials, showed no efficacy over placebo in theoverall population, and is being pursued in further testing only inwomen. Rifaximin (Salix Pharmaceuticals) has been studied for IBS-D,showing moderate activity, but there is significant concern for thedevelopment of antibiotic resistance and continued efficacy.

Latronex (alosetron, Prometheus Laboratories, Inc.) is the only drugapproved for IBS-D in the United States, albeit only for women. It hasno demonstrated analgesic properties. Importantly, it has a black boxwarning for serious adverse effects including, specifically, ischemiccolitis.

To date, no drug has been approved in the United States for chronic,unrestricted treatment of IBS-D.

Eluxadoline (Forest Laboratories, Inc.) is a μ opioid receptor agonistand 6 opioid receptor antagonist that has met primary endpoints ofimprovement of stool consistency and reduction of abdominal pain inPhase III testing. Its effect on pain reduction is modest at best andwithout a demonstrable effect on reducing colonic hypersensitivity thatresults in hyperalgesia. Moreover, several cases of pancreatitis, apotentially life threatening disease, were reported in Phase II trials.Cases of pancreatitis were reported even after patients with a knownhistory of biliary disease were excluded from clinical study enrollment.In general, μ agonists have a constricting effect on the Sphincter ofOddi, a muscular valve that regulates the flow of bile and pancreaticjuice from the bile duct into the duodenum. Buprenorphine, because ofits partial μ agonist effect and κ antagonist effect, does not result inincreased tone or constriction of the Sphincter of Oddi. We expect thatthe buprenorphine dimer, with the same receptor pharmacology asbuprenorphine, will also have no constricting effect on the Sphincter ofOddi.

There has accordingly been a long-standing need for a chronic treatmentof IBS-D that decreases intestinal motility, thereby decreasing theincidence of diarrhea, is an analgesic, is not associated withpancreatitis, and more than merely treating symptoms, potentiallyaddresses underlying hypersensitivity and resulting hyperalgesiaassociated with IBS-D.

BRIEF SUMMARY OF THE INVENTION

In one aspect, provided herein is a novel dimer comprising twobuprenorphine drug molecules, conjugated to each other by O-alkylationthrough their phenolic groups to yield the structure of Formula (I):

or a pharmaceutically acceptable salt or solvate thereof

In Formula (I), the compound is:2,2′-((4aR,4a′R,6S,6′S,7S,7′S,12bR,12b′R)-9,9′-(ethane-1,2-diylbis(oxy))bis(3-(cyclopropylmethyl)-7-methoxy-1,2,3,4,5,6,7,7a-octahydro-4a,7-ethano-4,12-methanobenzofuro[3,2-e]isoquinoline-9,6-diyl))bis(3,3-dimethylbutan-2-ol)(Compound 1, or buprenorphine dimer). Its molecular weight is 961.28.

Initially, we synthesized this buprenorphine dimer intending to achievea prodrug that would deter abuse. By dimerizing the parent compounds attheir phenolic hydrogen positions, we believed opioid activity would beabated pending first-pass metabolism in the liver to release the parentcompound. We believed that drug metabolism would be facilitated bycytochrome P-450 enzymes (CYP 3A4 and CYP2D6) when they interacted withthe methylene carbons attached to the phenolic oxygen molecules of thedimer. The resulting O-dealkylation would eventually releasebuprenorphine into systemic circulation.

Our expectations were supported by the literature regarding conjugationto phenolic hydrogen generally, albeit not with our particular linker.Conventional wisdom has been that derivatization of the phenolic groupsof morphinones by replacing hydrogen with a less hydrophilic substituentwould substantially reduce the opioid potency of the resulting opioid.See Feinberg Andrew F, et al. Proc Natl Acad Sci. USA Volume 73 no 11 p4215-4219 (1976). According to R. Richards, Opioid Analgesics(www.faculty.smu.edu): “A free phenol group is crucial for analgesicactivity.” In Anesth Analg 1984; 63; 143-51 at 145 et seq, D. H. Thorpesays “Another portion of the morphine molecule thought to interact withthe receptor is the phenol moiety. Muzzling the free hydroxyl group witha methyl group reduces potency more than ten-fold . . . . ” The authorgoes on to cite other studies showing that larger alkyl groups have aneven more deleterious effect, concluding that “bulkiness . . . isresponsible for the decreased binding effect.” See also U.S. Pat. Nos.8,183,376 and 8,461,171.

Despite these expectations, the resulting dimer turned out to havewholly unanticipated properties, unsuiting its utility as a prodrug butsuiting it for very different indications than those we firstenvisioned. The buprenorphine moieties, far from exhibiting the sterichindrance we anticipated, retained their characteristic opiate receptorpharmacology. The dimer proved resistant to enzymatic metabolism in thegastrointestinal milieu. It is also highly resistant to tampering, as byfree-basing. When orally administered it is essentially non-absorbed andso does not enter the systemic circulation. These serendipitousobservations led us to conceive new indications that were advantaged bythese properties, viz, peripheral analgesia in the GI tract.

The novel compound of the invention addresses a long-felt need for aneffective treatment of IBS-D that addresses both pain and diarrhea,reduces visceral (colonic) hypersensitivity, does not have the drawbacksof other agents utilized for this purpose and that can be prescribedsafely on a chronic basis. Accordingly, the invention comprises the useof the compound for the treatment of IBS-D, especially when visceralhypersensitivity and hyperalgesia are present. The compound may alsofind application as an adjuvant with other drugs such as teduglutide fortreating other intestinal conditions such as short bowel syndrome, tosafely reduce intestinal transit time. Pharmaceutical compositionsaccording to the invention comprise a buprenorphine dimer of Formula (I)formulated as an oral tablet or capsule, extended release oral tablet orcapsule, intramuscular or subcutaneous depot injection, or transdermalpatch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a bar chart illustrating the stability of buprenorphinedimer when exposed to CYP enzymes in the presence and absence of aco-factor.

FIG. 2 provides a bar graph showing the stability of buprenorphine dimerto aqueous conditions, as well as acidic and basic condition, each atroom temperature and at 140° F. for the indicated period of time.

FIG. 3 provides the results of buprenorphine dimer receptor bindingexperiments-μ receptor.

FIG. 4 provides the results of buprenorphine dimer receptor bindingexperiments-κ receptor.

FIG. 5 provides μ agonist functional assay results for the buprenorphinedimer.

FIG. 6 provides μ antagonist functional assay results for thebuprenorphine dimer.

FIG. 7 provides the results of oral and IV bioavailability forbuprenorphine and the buprenorphine dimer.

FIGS. 8 and 9 provide the graphs for stress-induced fecal output of maleCD-1 mice according to the evaluation of Example 7.

FIG. 10 demonstrates that buprenorphine dimer decreases fecal output ina dose-dependent manner.

FIG. 11 shows the effect of buprenorphine dimer on gastrointestinalmotility in post inflammatory models according to Example 7.

DETAILED DESCRIPTION OF THE INVENTION

The buprenorphine dimer of the invention exhibits the μ-receptor partialagonist and full κ-receptor antagonist activity characteristic of theparent buprenorphine. The κ-receptor antagonist activity is veryimportant since it appears to have multiple functions. As the receptoragonist dimer described herein also has a κ-receptor antagonist effect,the μ-receptor agonist reduction of intestinal motility is moderated,reducing the possibility of significant constipation. In addition,κ-receptor antagonist effect appears to prevent the constrictiveμ-receptor agonist effect on the smooth muscle tissue of the Sphincterof Oddi, avoiding the possibility of pancreatitis. In animal and humanstudies, it has been shown that buprenorphine also reduced colonic andskin hypersensitivity and hyperalgesia. Visceral hypersensitivity is animportant component of the etiology of IBS-D and we believe thatκ-receptor antagonism of the buprenorphine dimer will play an importantrole in symptom reduction. We have now also further demonstrated inanimal models that the described buprenorphine dimer, owing to itsconjoint retention of buprenorphine receptor pharmacology andnon-systemic absorption (i.e., no absorption from the intestinal tract),is suitable for the chronic treatment of IBS-D, while substantiallyeliminating the prospects for abuse. This surprising discovery indicatesthat the dimer described herein would be superior to available agentsthat are utilized in the treatment of IBS-D because it (1) addresses theunderlying visceral (colonic) hypersensitivity and consequenthyperalgesia characteristic of IBS-D, (2) will side-step the possibilityof pancreatitis associated with eluxadoline, (3) will mitigate diarrheawithout the risk of constipation; and (4) will achieve the foregoingwithout the troublesome systemic and central nervous system adverseeffects attributed to buprenorphine itself.

As noted above, the present invention provides a dimeric form ofbuprenorphine where the two buprenorphine molecules are linked via acovalent bond between the phenolic (3-hydroxyl) functional group of eachbuprenorphine molecule and an ethylene linker. The ethylene linkerserves as a spacer between the two buprenorphine molecules and isthought to prevent the two bulky buprenorphine molecules from adoptingan enclosed ring conformation via either a covalent, ionic or Van derWaal interaction between other functional groups on the molecules.

Surprisingly, when two drug molecules are conjugated to each other viaan ethylene spacer, wherein the spacer is attached to the phenyl ring ofeach drug molecule via an ether bond, the resulting dimer is found to bechemically and metabolically stable, and is not de-conjugated whenexposed to metabolic enzymes. Additionally, surprisingly andunexpectedly, the dimer retains the pharmacological activity of theparent compound, yet has negligible systemic exposure upon oraladministration.

In contrast to buprenorphine, the buprenorphine dimer, prepared asdescribed herein, is found to be not absorbed after oral administrationand furthermore retains the opioid μ and κ activity not only in form butalso direction, viz. neither receptor affinity nor activity iscompromised. Additionally, the buprenorphine dimer described herein isrelatively stable to metabolism in in vivo and in vitro experiments. Thebuprenorphine dimer appears metabolically stable, even after exposure tothe liver of live mice, following intravenous injection. Thus,surprisingly and unexpectedly, despite the loss of absorptive propertiesas manifested by lack of gastrointestinal absorption and metabolicinactivation, the opioid functionality of the dimer was not lost and itsgastrointestinal opioid effects were manifested as a decrease ofmotility and anti-diarrheal response after oral dosing. Thegastrointestinal effects were proportional to dose in mice models wherediarrhea (increased excreted pellets) is produced by a combination ofphysical and psychological stress. In another mouse model experiment inwhich the colon was sensitized by an inflammatory insult, the favorableresponse persisted beyond the acute period, three weeks after the acuteinsult, possibly indicating an effect of the dimer on decreasing thehypersensitivity and hyperalgesia of the mouse colonic membranes.

Still further, it was also found that the buprenorphine dimer retained,selectively, only the μ and the κ functions of buprenorphine, but wassignificantly stripped of its δ function. Stated differently, thebuprenorphine dimer, unlike buprenorphine, is a selective μ and κ activemolecule without significant δ activity.

Synthesis of the Buprenorphine Dimer

Synthesis of the buprenorphine dimer provided herein can proceed by ageneral O-alkylation reaction in an organic solvent (such as, e.g.,acetonitrile, DMF, DMSO, NMP, DCM, THF, 1,4-Dioxane) in the presence ofinorganic base (such as, e.g., sodium hydroxide, potassium carbonate,sodium carbonate, cesium carbonate, potassium bicarbonate and sodiumbicarbonate) or organic base (such as, e.g., triethylamine, Hunig'sbase, DMAP and pyridine) at room temperature or elevated temperature.Suitable alkylating agents that can be used include diiodo, dibromo,dichloro, ditosylate, dimesylate and ditriflate reagents (e.g.,1,2-ethylene ditosylate, 1,2-ethylene dimesylate). The free base or asalt of buprenorphine can be employed as a starting material in thesynthesis.

Pharmaceutical Compositions of the Dimer—General

In certain embodiments, provided herein are compositions comprising abuprenorphine dimer of Formula (I). A pharmaceutical composition canfurther comprise a pharmaceutically acceptable carrier. Illustrativepharmaceutically acceptable carriers and formulations are describedbelow. Such pharmaceutical compositions can be used to treatdiarrhea-predominant IBS.

As will be appreciated, a pharmaceutically acceptable salt of a dimermay be used instead of or in addition to a dimer in any or all of thecompositions and methods of treating discussed herein. Thus, in specificembodiments, a pharmaceutically acceptable salt of the dimer (i.e., anypharmaceutically acceptable salt of any of the dimers) is used in themethods of the invention. These salts can be prepared, for example, insitu during the final isolation and purification of the compound or byseparately reacting the purified compound in its free base form with asuitable organic or inorganic acid and isolating the salt thus formed.In some embodiments, the pharmaceutically acceptable salt of thebuprenorphine dimer is prepared using acetic, alginic, anthranilic,benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic,glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phenylacetic, phosphoric, propionic, salicylic, stearic, succinic,sulfanilic, sulfuric, tartaric acid, or p-toluenesulfonic acid. Forfurther description of pharmaceutically acceptable salts that can beused in the methods described herein see, for example, S. M. Berge etal., “Pharmaceutical Salts,” 1977, J. Pharm. Sci. 66:1-19, which isincorporated herein by reference in its entirety.

The buprenorphine dimer of the invention can exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like. In general, the solvated forms areconsidered equivalent to the unsolvated forms for the purposes of thepresent invention. In a specific embodiment, the solvated form of thedimer is a hydrate.

In general, salt formation may improve shelf life of the resultanttherapeutic agent. Appropriate salt synthesis can afford products thatare crystalline, less prone to oxidation and easy to handle. Varioussalts can be prepared that would afford stable and crystallinecompounds. A few examples are hydrochloric, sulfuric, p-toluenesulfonic,methanesulfonic, malonic, fumaric, and ascorbic acid salts.

In certain specific embodiments, such a pharmaceutical composition isformulated as oral tablet or capsule, extended release oral tablet orcapsule (hard gelatin capsule, soft gelatin capsule), sublingual tabletor film, or extended release sublingual tablet or film. Illustrativepharmaceutically acceptable carriers and formulations are described inmore detail below.

Methods of Treatment—General

In specific embodiments, provided herein is a method for the treatmentof Diarrhea-Predominant Irritable Bowel Syndrome by reducing diarrhea,pain and gut hypersensitivity and hyperalgesia in a patient comprisingoral administration of a therapeutically effective amount of the dimer.A therapeutically effective amount is an amount which yields anappreciable and beneficial effect in a statistically significant numberof patients. In certain embodiments, the patient is a mammal. In morespecific embodiments, the patient is a human. In certain specificembodiments, the patient is a domesticated mammal such as a dog, a cat,or a horse.

Pharmaceutical Compositions, Dosing and Routes of Administration

The IBS-D drug provided herein can be administered to a subject orallyin the conventional form of preparations, such as capsules,microcapsules, tablets, granules, powder, troches, pills, suppositories,oral suspensions, syrups, oral gels, sprays, solutions and emulsions.Suitable formulations can be prepared by methods commonly employed usingconventional, organic or inorganic additives, such as an excipient(e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose,talc, calcium phosphate or calcium carbonate), a binder (e.g.,cellulose, methylcellulose, hydroxymethylcellulose,polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic,polyethyleneglycol, sucrose or starch), a disintegrator (e.g., starch,carboxymethylcellulose, hydroxypropylstarch, low substitutedhydroxypropylcellulose, sodium bicarbonate, calcium phosphate or calciumcitrate), a lubricant (e.g., magnesium stearate, light anhydrous silicicacid, talc or sodium lauryl sulfate), a flavoring agent (e.g., citricacid, menthol, glycine or orange powder), a preservative (e.g, sodiumbenzoate, sodium bisulfite, methylparaben or propylparaben), astabilizer (e.g., citric acid, sodium citrate or acetic acid), asuspending agent (e.g., methylcellulose, polyvinyl pyrrolidone oraluminum stearate), a dispersing agent (e.g.,hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax(e.g., cocoa butter, white petrolatum or polyethylene glycol).

The dose of the buprenorphine dimer provided herein to be administeredto a patient is rather widely variable and can be subject to thejudgment of a health-care practitioner. Dosage may be properly varieddepending on the age, body weight and medical condition of the subjectand the type of administration. In one embodiment, one dose is given perday. In any given case, the amount of the dimer provided hereinadministered will depend on such factors as the solubility of the activecomponent, the formulation used and the route of administration. Theeffective amount of the buprenorphine dimer drug provided herein in thepharmaceutical composition will be at a level that will exercise thedesired effect, preferably, for example, about 0.15 mg/kg of an IBS-Dpatient's body weight to about 7.2 mg/kg of a patient's body weight,more preferably from about 0.7 mg/kg of an IBS-D patient's body weightto about 3.0 mg/kg of a patient's body weight, and still more preferablyabout 1.5 mg/kg of a patient's body weight in unit dosage for oraladministration. Alternatively, from about 10 to about 500 mg, preferablyfrom about 50 to about 200 mg, more preferably about 100 mg, will beadministered to an IBS-D patient.

The buprenorphine dimer provided herein can be administered, e.g., once,twice, or three times daily, preferably once per day. The dimer providedherein can be administered orally for reasons of convenience. In oneembodiment, when administered orally, the dimer provided herein isadministered with a meal and water. In another embodiment, the dimerprovided herein is dispersed in water or juice (e.g., apple juice ororange juice) and administered orally as a suspension.

Alternatively, the buprenorphine dimer provided herein can also beadministered rectally or by other transmucosal routes. The mode ofadministration is left to the discretion of the health-carepractitioner.

In one embodiment, provided herein are capsules containing the dimerwithout an additional carrier, excipient or vehicle.

In another embodiment, provided herein are compositions comprising aneffective amount of the dimer and a pharmaceutically acceptable carrieror vehicle, wherein a pharmaceutically acceptable carrier or vehicle cancomprise an excipient, diluent, or a mixture thereof.

The oral compositions can be in the form of tablets, chewable tablets,capsules, solutions, troches and suspensions and the like. Compositionscan be formulated to contain a daily dose, or a convenient fraction of adaily dose, in a dosage unit, which may be a single tablet or capsule orconvenient volume of a liquid. In one embodiment, the solutions areprepared from water-soluble salts, such as the hydrochloride salt. Ingeneral, all of the compositions are prepared according to known methodsin pharmaceutical chemistry. Capsules can be prepared by mixing a dimerprovided herein with a suitable carrier or diluent and filling theproper amount of the mixture into capsules. The usual carriers anddiluents include, but are not limited to, inert powdered substances suchas starch of many different kinds, powdered cellulose, especiallycrystalline and microcrystalline cellulose, sugars such as fructose,mannitol and sucrose, grain flours and similar edible powders.

Tablets can be prepared by direct compression, by wet granulation, or bydry granulation. Their formulations usually incorporate diluents,binders, lubricants and disintegrators as well as the compound. Typicaldiluents include, for example, various types of starch, lactose,mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such assodium chloride and powdered sugar. Powdered cellulose derivatives arealso useful. Typical tablet binders are substances such as starch,gelatin and sugars such as lactose, fructose, glucose and the like.Natural and synthetic gums are also convenient, including acacia,alginates, methylcellulose, polyvinylpyrrolidine and the like.Polyethylene glycol, ethylcellulose and waxes can also serve as binders.

A lubricant might be necessary in a tablet formulation to prevent thetablet and punches from sticking in the dye. The lubricant can be chosenfrom such slippery solids as talc, magnesium and calcium stearate,stearic acid and hydrogenated vegetable oils. Tablet disintegrators aresubstances that swell when wetted to break up the tablet and release thecompound. They include starches, clays, celluloses, algins and gums.More particularly, corn and potato starches, methylcellulose, agar,bentonite, wood cellulose, powdered natural sponge, cation-exchangeresins, alginic acid, guar gum, citrus pulp and carboxymethyl cellulose,for example, can be used as well as sodium lauryl sulfate. Tablets canbe coated with sugar as a flavor and sealant, or with film-formingprotecting agents to modify the dissolution properties of the tablet.The compositions can also be formulated as chewable tablets, forexample, by using substances such as mannitol in the formulation.

When it is desired to administer the drug provided herein as asuppository, typical bases can be used. Cocoa butter is a traditionalsuppository base, which can be modified by addition of waxes to raiseits melting point slightly. Water-miscible suppository bases comprising,particularly, polyethylene glycols of various molecular weights are inwide use.

The effect of the drug provided herein can be delayed or prolonged byproper formulation. For example, a slowly soluble pellet of the dimercan be prepared and incorporated in a tablet or capsule, or as aslow-release implantable device. The technique also includes makingpellets of several different dissolution rates and filling capsules witha mixture of the pellets. Tablets or capsules can be coated with a filmthat resists dissolution for a predictable period of time.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Synthesis

Buprenorphine dimer was synthesized as shown below.

Buprenorphine HCl-salt (5.0 g, 10.68 mmol, 1 equiv) and potassiumcarbonate (42.73 mmol, 4 equiv) were charged in a 3-neck round bottomflask followed by anhydrous DMSO (50 ml, 10 vol). The mixture was heatedto 60° C. and 1,2-dibromoethane (9.2 mL, 106.8 mmol, 10 equiv) was addedslowly. The reaction mixture was stirred at 60° C. for 16 h then cooledto room temperature, diluted with water and extracted withdichloromethane. The combined organic portions were washed with brine,dried (anhydrous Na₂SO₄), filtered and concentrated under reducedpressure to afford a viscous liquid. The crude product was purified bysilica gel chromatography using 0-5% MeOH/DCM to afford 4.2 g (69%)Intermediate 1 as off-white foamy solid.

Buprenorphine HCl-salt (1.74 g, 3.72 mmol) and potassium carbonate (2.0g, 14.87 mmol, 4 equiv) were charged in a 3-neck round bottom flaskfollowed by anhydrous DMSO (10 mL). The mixture was heated to 60° C. andIntermediate 1 (3 g, 5.22 mmol, 1.4 equiv) dissolved in 7 mL ofanhydrous DMSO was added dropwise over a period of 2 h. The reactionmixture was stirred at 60° C. for 16 h then cooled to room temperature,diluted with water and extracted with dichloromethane. The organic layerwas washed with brine, dried (anh. Na₂SO₄), filtered and concentratedunder reduced pressure to afford a viscous liquid. The crude product waspurified by silica gel chromatography using 0-5% MeOH/DCM to affordBuprenorphine dimer-FB (free base) as foamy solid (2.8 g, 77%).

5.5 g (5.7 mmol) of bi-conjugate (buprenorphine dimer-FB) was dissolvedin 50 mL of ethyl acetate at room temperature under nitrogen. 3.43 mL(6.9 mmol, 1.2 equiv) of 2N HCl in ether was added drop-wise at roomtemperature. The reaction mixture was stirred at room temperature foradditional hour and filtered to obtain a solid. The solid was furtherwashed with 100 ml of ethyl acetate and dried under vacuum to affordbuprenorphine dimer (bis HCl salt) as white solid (5.8 g, 98%). ¹H NMR(300 MHz, DMSO-d₆): δ 9.75 (br, 2H), 6.88 (d, J=9.2 Hz, 2H), 6.67 (d,J=9.2 Hz, 2H), 4.66 (s, 2H), 4.23-4.42 (m, 4H), 3.84-3.92 (m, 2H), 3.40(s, 6H), 3.21-3.35 (m, 5H), 2.98-3.20 (m, 7H), 2.64-2.85 (m, 4H),2.12-2.26 (m, 4H), 1.72-1.94 (m, 4H), 1.38-1.52 (m, 4H), 1.26 (s, 6H),0.99 (s, 20H), 0.48-0.76 (m, 10H), 0.32-0.42 (m, 4H); MS: m/z 962 (M+1)⁺

Example 2 Illustrative Pharmaceutical Compositions

The following composition can be used for an oral tablet ofBuprenorphine dimer.

TABLE 1 Ingredients % w/w Dimer 2 Lactose 83.6 Colloidal Silicon dioxide0.67 Microcrystalline cellulose 10 Croscarmellose sodium 3.4 Magnesiumstearate 0.33

Example 3 Assays In Vitro Assay Metabolic Stability of BuprenorphineDimer

Incubations of the dimer (e.g., 1 μM) with human liver microsomes (e.g.,1 mg protein/mL) was carried out using a Tecan Liquid Handling System(Tecan), or equivalent, at 37±1° C. in 0.2-mL incubation mixtures (finalvolume) containing potassium phosphate buffer (50 mM, pH 7.4), MgCl₂ (3mM) and EDTA (1 mM, pH 7.4) with and without a cofactor,NADPH-generating system, at the final concentrations indicated in a96-well plate format. The NADPH-generating system consisted of NADP (1mM, pH 7.4), glucose-6-phosphate (5 mM, pH 7.4) and glucose-6-phosphatedehydrogenase (1 Unit/mL). The dimer was dissolved in aqueous methanolicsolution (methanol 0.5% v/v, or less). Reactions were started typicallyby addition of the cofactor, and stopped at four designated time points(e.g., up to 120 min) by the addition of an equal volume of stop reagent(e.g., acetonitrile, 0.2 mL containing an internal standard). Zero-timeincubations served as 100% value to determine percent loss of substrate.Incubations were carried out in triplicate with an exception forzero-time samples (which were incubated in quadruplicate). Zero-cofactor(no NADPH) incubations were performed at zero-time and the longest timepoint. The samples were subjected to centrifugation (e.g., 920×g for 10min at 10° C.) and the supernatant fractions analyzed by LC-MS/MS.Additional incubations were carried out with microsomes in which werereplaced with a marker substrate (e.g., dextromethorphan to monitorsubstrate loss) as positive controls to determine if the test system ismetabolically competent.

The above samples were analyzed by LC-MS/MS. Analysis was performed forthe samples at each incubation solution. Results were determined by acomparison of peak ratios over the time course of the experiment(typically reported as “% Parent Remaining”).

Data were calculated with a LIMS (includes Galileo, Thermo FisherScientific Inc. and reporting tool, Crystal Reports, SAP), thespreadsheet computer program Microsoft Excel (Microsoft Corp.) orequivalent. The amount of unchanged parent compound will be estimated(to determine approximate percent substrate remaining in eachincubation) based on analyte/internal standard (IS) peak-area ratiosusing a LIMS, Analyst Instrument Control and Data Processing Software(AB SCIEX), or equivalent.

Results: Results as shown in FIG. 1 indicate that the dimer ofbuprenorphine was relatively stable in presence of microsomal enzymesfor the duration of the assay. The microsomal enzymes are typicallyresponsible for metabolism of drugs such as buprenorphine. The dimer wasstable in presence of the microsomes, with or without the co-factor. Theassay was terminated at 2 hours as enzymes are typically not stablebeyond 2 hours at incubation temperatures of 37° C.

Stability Assay

The goal of the laboratory-based studies was to evaluate the ease withwhich the patient can retrieve buprenorphine from the dimer and thuscompromise its abuse deterrent properties.

These studies facilitate the understanding of the ease with which apotential abuser could cleave the dimer using household chemicals suchas baking soda, acid or simple heating in water. Buprenorphine dimerstability was assessed at room temperature in untreated tap water and inpresence of acid (1N HCl) or base (5% aqueous sodium bicarbonate). Thedimer was relatively stable under those conditions and under theseconditions did not appreciably degrade to buprenorphine. See FIG. 2.

Results: As shown in FIG. 2, the buprenorphine dimer remained stable anddid not degrade to release buprenorphine either at room temperature orelevated temperature under extreme pH conditions even as long as 30minutes.

These studies also facilitate the understanding of the stability of thedimer in the gastrointestinal tract, which exhibits a gradient pH alongits length in both IBS-D and healthy patients. The pH ranges from 1 dueto excretion of hydrochloric acid from the parietal cells of the stomachto 8 in the colon. The proximal portion of the gastrointestinal tract ismost acidic where the distal end is the least acidic.

Example 4 Receptor Binding Activity

This example illustrates the binding of the buprenorphine dimer providedherein to the following receptors: μ-opioid receptor; κ-opioid receptor;and δ-opioid receptor.

Human μ Opioid Receptor Binding Assay

Membranes from Chinese Hamster Ovary cells expressing the human μ opioidreceptor (Perkin Elmer #RBHOMM400UA) were homogenized in assay buffer(50 mM Tris, pH 7.5 with 5 mM MgCl2) using glass tissue grinder, Teflonpestle and Steadfast Stirrer (Fisher Scientific). The concentrates ofthe membranes were adjusted to 300 μg/mL in assay plate, a 96 well roundbottom polypropylene plate. The compound to be tested was solubilized inDMSO (Pierce), 10 mM, then diluted in assay buffer to 3.6 nM. In asecond 96 well round bottom polypropylene plate, known as the premixplate, 60 μL of 6× compound was combined with 60 μL of 3.6 nM³H-Nalaxone. From the premix plate 50 μL was transferred to an assayplate containing the membranes, in duplicate. The assay plate wasincubated for 2 h at room temperature. A GF/C 96 well filter plate(Perkin Elmer #6005174) was pretreated with 0.3% polyethylenimine for 30min. The contents of the assay plate were filtered through the filterplate using a Packard Filtermate Harvester, and washed 3 times with 0.9%saline at 4° C. The filter plate was dried, underside sealed, and 304,Microscint 20 (Packard #6013621) was added to each well. A Topcount-NXTMicroplate Scintillation Counter (Packard) was used to measure emittedenergies in the range of 2.9 to 35 KeV. Results were compared to maximumbinding, wells receiving no inhibitions. Nonspecific binding wasdetermined in presence of 50 μM unlabeled naloxone. The biologicalactivity of the dimer is shown in FIG. 3.

Results: The graphs in FIG. 3 show that the dimer has significantaffinity for the opioid μ receptor The opioid μ receptor affinity of thebuprenorphine dimer at 10⁻⁸M (˜10 ng) and the profile was similar tothat of buprenorphine.

Human κ Opioid Receptor Binding Assay

Membranes from cloned HEK-293 cells expressing the human kappa opioidreceptor (Amersham Biosciences UK Ltd. 6110558 200U) were homogenized inassay buffer (50 mM Tris, pH 7.5 with 5 mM MgCl2) using glass tissuegrinder, Teflon pestle and Steadfast Stirrer (Fisher Scientific). Theconcentrates of the membranes were adjusted to 300 μg/mL in assay plate,a 96 well round bottom polypropylene plate. The compound to be testedwas solubilized in DMSO (Pierce), 10 mM, then diluted in assay buffer to3.6 nM. In a second 96 well round bottom polypropylene plate, known asthe premix plate, 60 μL of 6× compound was combined with 60 μL of 3.6 nM³H-Diprenorphine (DPN). From the premix plate, 50 μL was transferred toan assay plate containing the membranes, in duplicate. The assay platewas incubated for 18 h at room temperature. A GF/C 96 well filter plate(Perkin Elmer #6005174) was pretreated with 0.3% polyethylenimine for 30min. The contents of the assay plate were filtered through the filterplate using a Packard Filtermate Harvester, and washed 3 times with 0.9%saline at 4° C. The filter plate was dried, underside sealed, and 30 μLMicroscint 20 (Packard #6013621) was added to each well. A Topcount-NXTMicroplate Scintillation Counter (Packard) was used to measure emittedenergies in the range of 2.9 to 35 KeV. Results were compared to maximumbinding, wells receiving no inhibitions. Nonspecific binding wasdetermined in presence of 50 μM unlabeled naloxone. The biologicalactivity of the dimer is shown in FIG. 4.

Results: FIG. 4 describes the opioid κ receptor agonist profile of thebuprenorphine monomer and the dimer. Neither the monomer nor the dimerof buprenorphine has lost its affinity for the κ receptor.Qualitatively, as with buprenorphine, the binding of the buprenorphinedimer to opioid κ receptor increases with concentration. It is estimatedthat at about 1 μg, the profile of the opioid κ receptor affinity of thedimer was similar to that of buprenorphine.

Human δ Opioid Receptor Binding Assay

The assay was designed to test the ability of a compound to interferewith the binding of tritiated naltrindole to the human δ subtype 2opioid receptor. Membranes from Chinese Hamster Ovary cells expressingthe human δ subtype 2 opioid receptor (Perkin Elmer #RBHODM400UA) werehomogenized in assay buffer (50 mM Tris, pH 7.5 with 5 mM MgCl₂) using aglass tissue grinder, Teflon pestle and Steadfast Stirrer (FisherScientific). The concentration of membranes was adjusted to 100 μg/mL inan assay plate, a 96 well round bottom polypropylene plate. The compoundto be tested was solubilized in DMSO, 10 mM, then diluted in assaybuffer to 6× the desired final concentration. The ligand, ³H-natrindole(Perkin Elmer #NET-1065) was also diluted in assay buffer to 6 nM.Aliquots of ³H-natrindole (50 μL) were transferred to the assay platecontaining the membranes in duplicate. The assay plate was incubated for30 minutes at room temperature. A GF/C 96 well filter plate (PerkinElmer #6005174) was pretreated with 0.3% polyethylenimine for 30 min.The contents of the assay plate were filtered through the filter plateusing a Packard Filtermate Harvester, and washed 3 times with 0.9%saline at 4° C. The filter plate was dried, the underside sealed, and a30 tit MictoS=scint 20 (Packard #6013621) added to each well. ATopcount-NXT Microplate Scintillation Counter (Packard) was used tomeasure emitted energies in the range of 2.9 to 35 KeV. Results werecompared to maximum binding, wells receiving no inhibitors. Nonspecificbinding was determined in the presence of 1 μM unlabelled Natrindole.The biological activity of the buprenorphine dimer is shown in Table 2below.

TABLE 2 Compound IC50 Ki Buprenorphine dimer 7.6 nM 2.87 nM

Relative to the μ and κ opioid receptors, the dimer had poor affinityfor the δ receptor.

Example 5 Receptor Stimulation Activity

This example illustrates the ability of the buprenorphine dimer compoundprovided herein to stimulate the μ-opioid receptor-mediated signaling.

μ Opioid Receptor Agonist and Antagonist Functional Assays: [³⁵S]GTPγSBinding Assay in Chinese Hamster Ovaries Expressing Human μ Receptors(CHO-hMOR) Cell Membranes

Briefly, CHO-hMOR cell membranes were purchased from Receptor BiologyInc. (Baltimore Md.). About 10 mg/ml of membrane protein was suspendedin 10 mM TRIS-HCl pH 7.2, 2 mM EDTA, 10% sucrose, and the suspensionkept on ice. One mL of membranes was added to 15 mL cold binding assaybuffer containing 50 mM HEPES, pH 7.6, 5 mM MgCl₂, 100 mM NaCl, 1 mM DTTand 1 mM EDTA. The membrane suspension was homogenized with a polytronand centrifuged at 3000 rpm for 10 min. The supernatant was donecentrifuged at 18,000 rpm for 20 min. The pellet was resuspended in 10ml assay buffer with a polytron.

The membranes were pre-incubated with wheat germ agglutinin coated SPAbeads (Amersham) at 25° C., for 45 min in the assay buffer. The SPA bead(5 mg/ml) coupled with membranes (10 μg/ml) were then incubated with 0.5nM [³⁵S]GTPγS in the assay buffer. The basal binding was that takingplace in the absence of added test compound; this unmodulated bindingwas considered as 100%, with agonist stimulated binding rising to levelssignificantly above this value. A range of concentrations of receptoragonist SNC80 was used to stimulate [³⁵S]GTPγS binding. Both basal andnon-specific binding were tested in the absence of agonist; non-specificbinding determination included 10 μM unlabeled GTPγS.

Buprenorphine dimer was tested for function as an antagonist byevaluating its potential to inhibit agonist-stimulated GTPγS bindingusing D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) as the standard.Radioactivity was quantified on a Packard Top Count. The followingparameters were calculated:

% Stimulation=[(test compound cpm−non-specific cpm)/(basalcpm−non-specific cpm)]*100%

Inhibition=(% stimulation by 1 μM SNC80-% stimulation by 1 μM SNC80 inpresence of test compound)*100/(% stimulation by 1 μM SNC80-100).

EC₅₀ was calculated using GraphPad Prism. A graph for the compoundtested is shown in FIGS. 5 and 6.

Results: Data shown in FIG. 5 indicates that the dimer is a potent μagonist. The results also indicate that the opioid μ receptor activityof the dimer at 10⁻⁶M (˜1 μg) is similar to that of buprenorphine. Datain FIG. 6 shows that the dimer does not function as a μ-antagonist.

Example 6 In Vivo Pharmacokinetic Study

The animal pharmacokinetic studies were conducted at John HopkinsMedical Institute. Animals used were CD-1 mice (about 35 gms, n=3 pertime point). Drugs tested were buprenorphine and buprenorphine dimer.Dose 10 mg/kg IV and oral gavage. Blood collected at time 0, 30 min and1, 2, 6 and 24 hours. Blood samples for the drug were analyzed afterharvesting the plasma and by LC/MS/MS as follows:

A standard curve was prepared in mouse plasma spiked with the test drug(10-25000 nM). Plasma samples (50 μL) were extracted in 300 μL,acetonitrile containing losartan or buprenorphine-d₄ as internalstandard. Extracts were centrifuged at 16000×g at 4° C. for 5 minutes.Supernatants (250 μL) were transferred to a new tube and dried under N₂at 45° C. for 1 hour. Samples were reconstituted with 100 μL of 30%acetonitrile, vortexed and centrifuged. Supernatants (90 μL) weretransferred to LC vials and 10 μL was injected on LC/MS. See FIG. 7.

Results: FIG. 7 depicts the plasma concentration profiles of thebuprenorphine dimer after 10 mg oral and IV dose. The graph indicatesthat the absolute bioavailability, measured as a ratio of the area underthe concentration curve after oral and IV dose, of the dimer was 1% orless, where as that of the monomer was about 30%.

Example 7 In Vivo Assay Stress-Induced Fecal Output

The animals used in the studies are male CD-1 mice, average weight about30 to 35 g, with an average of 5 mice per dose group. The mice weregenerally housed in colony housing where they are housed 3 per cage inpolycarbonate cages with access to food and water ad lib. On the day ofthe experiment they are transported to the procedure room where theywere individually housed in 20 cm wide×20 cm deep×15 cm tall cages,equipped with a wire mesh bottom after intragastric administration oftest compound. During the test the animals were allowed access to waterad lib. The wire mesh bottomed tall cage creates a novel environmentwhich induces stress in mice. The number of pellets excreted wasdetermined on hourly basis. Results are shown in FIG. 8-10.

Results: FIGS. 8 and 9 show that oral doses of the buprenorphine dimersignificantly reduced the fecal output in mice versus placebo (vehicle).The doses investigated were 25 and 50 mg per kg. The results did notchange even when the animals with zero fecal output, suggesting extremesensitivity, were removed from the analysis. FIG. 10 shows that fecaloutput in mice decreases with dose, which indicates a truepharmacological effect.

In Vivo Assay Effect on Post-Inflammatory Altered GI Transit Time

This test was designed to measure the effect of test substance ongastrointestinal hypersensitivity that occurs following inflammation.Post-inflammatory altered GI transit was induced in male CD-1 mice byinjecting freshly opened oil of mustard (95% pure allyl isothiocyanate,0.5% in ethanol). The effect of stress on GI is motility is evaluated 3to 4 weeks later, when although there is no longer inflammation, the GItract remains in a hypersensitive state. Effect of test substance wasmeasured after oral administration (intragastric gavage) and subjectingthe animals to environmental stress by housing them in a 20 cm wide×20cm deep×15 cm tall cages, equipped with a wire mesh bottom. During thetest, the animals were allowed access to water ad lib. The wire meshbottomed tall cage creates a novel environment, which induces stress inmice. The number of pellets excreted was determined on hourly totwo-hourly basis. See FIG. 11.

As shown in FIG. 11, the buprenorphine dimer at 25 mg per kgsignificantly decreased gastrointestinal motility, as measured by fecaloutput in post-inflammatory models. The graph also shows the increase infecal output in the mice not treated with mustard oil was transient anddid not last beyond 1 hour. The increased fecal output in mustard oiltreated animals persisted even at 2 hours. The dimer continued tocontrol gastrointestinal motility even at 2 hours and results werestatistically significant.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. To the extent there is conflict betweenthe priority applications and the present application, anyinconsistencies are to be resolved in favor of the present application.All publications and patents cited herein are hereby incorporated byreference in their entirety for all purposes.

What is claimed is:
 1. A buprenorphine dimer compound having Formula(I):

or a pharmaceutically acceptable salt or solvate thereof.
 2. Thebuprenorphine dimer compound of claim 1, wherein the compound is in theform of a pharmaceutically acceptable salt.
 3. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier orexcipient and a buprenorphine dimer compound of claim
 1. 4. A method oftreating or managing gastrointestinal hyperalgesia wherein the methodcomprises administering to a patient in need thereof a therapeuticallyeffective amount of a buprenorphine dimer compound of claim 1
 5. Amethod of treating IBS-D in a patient in need thereof, said methodcomprising administering to said patient a therapeutically effectiveamount of a buprenorphine dimer compound of claim
 1. 6. A method inaccordance with claim 5, wherein the patient is a human and wherein thedose administered is about 0.1 mg/kg of the patient's body weight toabout 7.2 mg/kg of the patient's body weight
 7. A method in accordancewith claim 5, wherein the patient is a human and wherein the doseadministered is about 10 to about 50 mg daily.